Ebola viruses (EBOV) and Marburg virus (MARV) are two genera of enveloped viruses that constitute the family of Filoviridae. They are among the most lethal human pathogens, causing a severe hemorrhagic fever with mortality rates up to 90% for EBOV-Zaire. No vaccine or specific antiviral is available for human use. We propose to apply the results of our fundamental research to the development of a new antiviral strategy for filovirus infections. The proposal is based on our recent discovery that attachment of a cholesterol group to a peptide fusion inhibitor yields 3 major advantages: (1) increased potency, (2) localization of the peptide with the virus at the site of fusion activation, and (3) enhanced pharmacokinetic properties. By combining sequence optimization with cholesterol tagging, we propose to develop highly effective peptide fusion antivirals that inhibit filoviruses in vitro, and to test their therapeutic potential in a relevant animal model of filovirus infection. These fusion/entry inhibitory peptides will be designed to follow the virus to the site of fusion with the cell membrane, thus overcoming the major obstacle to the use of peptide inhibitors for viruses that fuse in intracellular compartments. We propose to develop filovirus fusion inhibitors by: Aim 1. Design and test filovirus fusion inhibitory peptides that are targeted to the plasma membrane, using (a) biophysical analysis-guided sequence optimization to enhance peptide interaction with the target sequence, and (b) membrane targeting to the site of fusion activation. Aim 2. Assess the effectiveness of the fusion inhibitors at protecting against EBOV infection in a murine model of lethal infection. We will determine (a) bioavailability of the targeted peptides, and (b) efficacy in challenge experiments. We will thereby obtain proof of principle for efficacy, allowing us to select peptides for advancement that have the most in vivo potential. The overarching high impact of this proposal lies in the proof-of-concept that will be established for enveloped viruses that fuse in the endosome. The new strategy would overcome the barrier to use of peptide antivirals for viruses that fuse in intracellular compartments, including orhomyxoviruses (influenza), flaviruses (West Nile virus, Dengue virus), coronaviruses (severe acute respiratory syndrome), arenaviruses (Junin, Lassa), all sharing a similar fusion machinery.